The use of target immobilised NMR screening to identify and develop fragment binders to Hsp90

Transcription

1 The use of target immobilised MR screening to identify and develop fragment binders to sp90 ot topics in drug discovery: finding the next lead 11 ovember 2009 John Porter 2009 UCB Celltech

2 Introduction 2 Fragment based drug design (FBDD) is becoming a popular method of finding starting points for drug discovery programmes Wanted to evaluate FBDD in-house Key issue is the choice of screening method to identify fragment binders ne such method is target immobilised MR screening (TIS). Review our experiences in evaluating TIS to find fragment binders to sp90 Comparison with other screening methods Some of the approaches that we have followed to develop the identified fragment hits

3 3 Fragment screening

4 Fragment screening 4 Libraries are typically smaller as fragment chemical space is smaller Bind with high atom efficiency but with low affinity Greater hit rate Typically require biophysical screening methods Ideally require structural information on how fragments bind for rapid progression

5 Why sp90 to validate fragment screening? 5 Protein is well-expressed (>100 mg/l) Crystallises readily >80 crystal structures in PDB MR solution structure solved Precedented target for fragment screening (positive controls available)

6 eat Shock Protein 90 (sp90) SP90 is an ATP-dependent molecular chaperone Responsible for conformation and stability of many oncogenic client proteins e.g. RAF, ErbB2, AKT Mutant oncoproteins particularly reliant on SP90 e.g. B-RAF, EGFR, KIT Many of these proteins are key for driving cancer phenotype Inhibition of sp90 leads to ubiquitination and degradation of client proteins 6 Potential one step combinatorial treatment for cancer terminal domain ucleotide & drug binding Middle domain Client protein binding and catalytic loop C-terminal domain Dimerization

7 Mode of action 7 ascent client protein delivered by sp70 Co-chaperones and partner proteins ATP Intermediate chaperone complex Inhibitor ADP ATP -P i Mature protein Mature chaperone complex Protein ubiquitination and degradation

8 8 sp90 Structure pdb:1amw Prodromou et al, Cell, 90, 65-70, 1997

9 Representative sp90 Inhibitors 9 2 I S R Me Me 2 Geldanamycin Analogues Me 17 Et Cl Radicicol analogues 2 CF 3 CF2024 VER (VP-AUY922) SX2112

10 Fragment Screening Deck 3 components Commercially sourced Selected by virtual screening and medicinal chemists (kinase focused) In house fragments Rule of 3 criteria MW <300 Da logp <3 BD 3 BA 3 Rotatable bonds <3 PSA <60 Å 2 o reactive or toxic functionality Screened for solubility QC by LC-MS Consists of 2389 compounds 10

11 Fragment Deck: Biochemical Assay 11 SP90 Colorimetric ATPase assay: tolerant to DMS but lack of sensitivity and colour interference with some fragments SP90 MG ATP ADP + P i MG 2389 Fragments Initial hits 94 Fragments I it-rate= 1.5% 94 hits > 10mM, n=1 it confirmation 37 Fragments 37 hits > 10mM, average, n=2

12 SP90 Biochemical screening assays SP90 fluorescence polarization competition binding assay VER UCB UCB Polarized light SP90 Cl Me 2 C F CEt S Me Me Polarized light ATP-ase activity IC 50 (μm) SP90 FP activity IC 50 (μm) F ITC K D (μm) nd 2 10 mm mm I 12 Fluorescence polarization competition assay is able to generate IC 50 s for fragments (~1mM limit)

13 STD MR 13 Wanted to compare biochemical screening with biophysical methods Chose Saturation Transfer Difference MR Relatively low throughput method requires pre-screening of fragment library Asp Structures Dock using GLD Check for key interactions ydrophobic pocket 438 Structures Visual Inspection Thr 184 Solvent accessible 132 Fragments Gly 97 Lys Fragments from Biochemical Assay 153 Fragments Selected

14 Fragment screening by STD MR spectroscopy Analysis of data is subjective (the magnitude of the STD effect as reflected in the S/ of the response) and time consuming Total hits 46 (30% hit rate) Includes 6 fragments identified from biochemical assay btained ligand/protein crystal structures for 5 hits UCB UCB Me S S 2 2 Binding Site FP Assay IC 50 (um) Ligand Efficiency A A 5mM - 14 UCB S S 2 A nd - Me UCB Me 2 C B UCB B nd -

15 Two binding sites identified 15 Me Site A S S S Site B S Site B Phe 138 Me 2 C Me 2 Met 98 Site A Asp 93 Green: Protein conformation for site A binders; Gold: Protein conformation for site B binders

16 Lessons learned from in-house screening 16 ~150 compounds were made from these starting points but no improvement in binding affinity Focused on the 5 binders for which structures were available ot enough diversity in starting point structures Biochemical assay too insensitive Structural information important Looked for an alternative source of finding fragment starting points

17 Target Immobilised MR Screening (TIS) 17 Zobio BV Based at The University of Leiden, The etherlands ffer comprehensive fragment screening service using TIS technology Uses a library of ~1400 diverse, commercially available compounds (co-developed with Pyxis Discovery, Delft, The etherlands) Every compound is aqueously 500 µm Every compound has QC 1 MR spectra recorded by ZoBio Conforms to commonly accepted fragment criteria

18 Target Immobilised MR Screening (TIS) 18 Protein immobilised on resin and packed into flow cell in MR spectrometer Library of fragments (in pools of 4-8 compounds) flows over protein and reference protein (P domain of Akt) Spectra acquired and processed to identify binders (reduction of signal) Reference protein avoids false positives Binder on-binder

19 The TIS Screening Station 19 Flow-injection probe capable of holding 2 samples of solid support simultaneously.

20 sp90 TIS Pilot Screen 20 ID # TIS WaterLGSY FP Activity IC 50 (μm) p 7.4 UCB Yes D 952 >5 mm UCB Yes D 160 >5 mm UCB * Yes D 322 >1 mg/ml UCB * Weak Weak D >1 mg/ml Structure Me 2 C S Me 2 Me Me UCB * Yes Yes 1714 >1 mg/ml UCB o o STD MR it >5 mm 2 * Aboul-ela et al, AACR-CI-ERTC Molecular Targets and Cancer Therapeutics, ovember 17-21, 2003, Boston, USA, Abstract A8 D ot determined

21 21 TIS screening of positive controls Binding S 2 on-binding 2

22 Results Confirmation that TIS identifies both sp90 binding sites 1393 compounds screened 3-4 weeks to complete (screening & data analysis) 91 hits (>35% difference in signal intensity) 6.5% hit rate 22 TIS sp90 STD MR sp90 Biochemical Fragment Screen sp90 Biochemical TS Corporate Library sp90 TIS Kinase TIS PPI-1 TIS PPI-2 its Compounds screened ~ it Rate 6.5% 31% 1.5% % 3.8% 7.4% 5.1%

23 sp90 TIS Workflow 23 Biochemical screen TIS hit ID Choose hits for validation TIS hit validation Purchase hits Docking and tractability assessment using binding model 29 hits selected for competition screen with VER and UCB (Site B) All appeared competitive 58 hits (39%) deemed tractable 53 compounds acquired X-ray crystallography KEY to chemistry progression Rank binders MR studies Confirmation of ATP site binders Chemistry strategy to grow fragments Lead series Medicinal chemistry begins

24 Crystallography TIS hits screened in crystallography soaking experiments 17 ligand/protein crystal structures obtained (< 2Å) Crystallisation success rate 35% 6 Site A binders and 11 Site B binders Attempts it Rate Site A Site B ZB hits 53 32% 6 11 UCB hits % 3 2 rthogonal screening method to prioritise TIS hits for focused crystallography? SQC MR SPR ITC

25 Crystallography its 25 Cmpd C 2 Me 2 C 2 Et Ph Me Crystal Structure Binding Site B B B SQC Kd mm (s.d) 2.75 (1.56) 2.65 (0.57) 11.7 (1.97) Bioassay IC 50 (μm) Mol wt Ligand efficiency i/a i/a i/a Me B nd i/a S B 8.89 (4.15) i/a A 7.11 (4.46) i/a

26 Crystallography its 26 Cmpd S Ph Me Me Et SEt Crystal Structure Binding Site SQC Kd mm (s.d) Bioassay IC 50 (μm) Mol wt Ligand efficiency* B nd i/a A A A A 0.6 (0.085) (0.032) (0.034) (0.014) i/a i/a Cl B nd i/a 197 -

27 Strategies for developing fragment hits Grow towards site B Grow towards site A Connect site A with site B via suitable linker verlay different site A (or B) binders 27

28 Rational design: growing from Site A to Site B SQC Kd 0.6 mm LE 0.48 Site B Phe 138 Cl Phe 138 Me CEt VER49009 Met 98 Met 98 Asp 93 Asp 93

29 Rational design: growing from Site A to Site B 29 Cmpd 8 UCB UCB C 2 C FP activity Ligand Mol Wt IC 50 (μm) Efficiency 5mM UCB UCB mM 251

30 Rational design: growing from Site A to Site B 30 2 IC μm Site B LE Site C Phe 138 Met 98 Asp 93

31 31 Rational design: growing from Site A to Site B Phe 138

32 Rational design: growing from Site A to Site B 32 Cmpd UCB UCB F FP activity IC 50 (μm) Mol Wt Ligand Efficiency CEt UCB

33 Rational design: growing from Site A to Site B Site B Phe138 Asp93 IC μm LE IC μm LE 0.239

34 34 Rational design: growing from Site A to Site B and beyond? Site B Site C

35 Fragment development: Analoguing 35 Met 98 Met 98 Phe 138 S 2 FP IC um Eff FP IC um LE Asp 93 2 Cl FP IC 50 inactive

36 Fragment development: Analoguing Cmpd UCB UCB UCB UCB Cl Cl Cl 2 FP IC 50 (μm) Mol Wt Ligand Efficiency S Me S Me Me Cl UCB S Cl UCB S Cl UCB S Cl UCB S

37 Fragment development: Analoguing Conformation A Lys 112 Site B Cl 2 Phe 138 S FP IC 50 8 um Eff Lys 112 Site B 37 Conformation B Phe 138 Met 98 Met 98 Asp 93 Asp 93

38 Summary 38 A number of methods for finding sp90 fragment binders have been explored TIS methodology has been validated Structural information drives understanding of binding modes Crystallography is not always successful-ideally need orthogonal methods Don t overlook analoguing! Potential medicinal chemistry starting points identified This target is no longer being pursued because of strategic changes

39 Acknowledgements 39 Alison Maloney Uye Udofoyo Gregg Siegal Mark Calmiano Kelly le Riche Rob eetebrij James Reuberson Rich Taylor Tom Ceska Jean Delgado Ian Whitcombe Colin Stubberfield Dave McMillan